A need exists for determining the molecular mass of high molecular weight organic molecules such as nucleic acids, proteins, oligosaccarides, and like moieties having molecular weights of 3000 daltons (Da) and more, and for polymer size determinations. Presently no accurate general method for such determinations exist.
Heretofore, the best known method for the determination of protein and nucleic acid masses is gel electrophoresis which at best has an accuracy of .+-.5%. Presently, the only method known for determining polymer size distribution is a gel permeation method which is recognized as imprecise and only measures relative sizes. More accurate mass spectrometric methods have been reported recently for protein mass determination, but this approach has not been extended to other polymers.
Mass spectrometric analysis of massive biopolymers such as nucleic acids, proteins, and oligosaccharides requires a means of volatilizing the molecules without fragmentation or degradation, or with controlled fragmentation, together with a means of ionizing the gas-phase molecules efficiently, again without inducing fragmentation. Slow heating of such molecules typically results in pyrolysis rather than volatilization. Thus, a number of desorption techniques have been developed which involve a very rapid input of energy into the target material, either by fast (mega-electron volt) or slow (kilo-electron volt) heavy-ion impact or by photon irradiation, to achieve desorption in a time that precludes complete degradation. Advantages are derived from dissolving the sample to be volatilized in a liquid or solid matrix, which, in the case of kilo-electron volt ion impact desorption, can act to minimize ion beam damage, or, for pulsed laser desorption, can serve as a chromophore, efficiently coupling the radiative energy into the material to be volatilized.
The present invention represents a substantial improvement over the prior art by determining molecular masses through the use of pulsed laser ablation, multiphoton ionization and time of flight mass spectrometry.